Medical compositions for intravesical treatment of bladder cancer

ABSTRACT

Anti-cancer coating compositions comprising 3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol (E09) are disclosed. More specifically, the coating compositions comprise EO9 and a formulation vehicle. The formulation vehicle improves the solubility and stability of EO9. Additionally, the coating compositions can include coating agents that provide better adhesion of the coating composition to the bladder wall during intravesical delivery of the coating composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/344,446, filed Nov. 1, 2001, and whose entire contents are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Bladder cancer accounts for approximately 2% of all malignant cancersand is the fifth and tenth most common cancer in men and women,respectively. The American Cancer Society estimated that 54,500 newcases and 11,700 deaths would have occurred in 1997. Superficial bladdercancers (pTa, pT1 and CIS) account for 70-80% of cancers at firstpresentation. Management of superficial bladder cancer may be achievedby endoscopic surgical resection often followed by a course of adjuvantintravesical chemotherapy or immunotherapy with the aim of botheradicating remaining tumor cells and preventing tumor recurrence (HerrH W (1987) Intravesical therapy—a critical review. Urol Clin N Am14:399-404). Both anti-neoplastics (Mitomycin C [MMC], epirubicin andthioTEPA) and immunotherapy (BCG) administered intravesically areeffective at reducing tumor recurrence rates although it is unclearwhether disease progression to muscle invasive tumors is prevented(Newling D (1990) Intravesical therapy in the management of superficialtransitional cell carcinoma of the bladder: the experience of the EORTCGU group, Br J Cancer 61:497-499; Oosterlink et al. (1993) A prospectiveEuropean Organization for Research and Treatment of Cancer GenitourinaryGroup randomized trial comparing transurethral resection followed by asingle instillation of epirubicin or water in single stage Ta, T1papillary carcinoma of the bladder. J Urol 149:749-752). Thisobservation in conjunction with the fact that mortality from bladdercancer is still high underscores the need to develop more effectivetherapeutic agents (Oosterlink et al. 1993).

One such therapeutic agent is MMC which belongs to a class of compoundsknown as bioreductive drugs (Workman 1994). MMC represents one of theantineoplastic agents used to treat superficial bladder cancers(Maffezzini et al, 1996, Tolley et al, 1996). MMC is activated to acytotoxic species by cellular reductases although the role of specificreductase enzymes involved in bioreductive activation remains poorlydefined and controversial (Cummings et al, 1998a). This is particularlytrue for the enzyme NQO1 (NAD(P)H:Quinone oxidoreductase, EC 1.6.99.2)which is a cytosolic flavoprotein which catalyses the two electronreduction of various quinone based compounds using either NADH or NADPHas electron donors (Schlager and Powis, 1988, Siegel et al, 1990). Thestructurally related compound E09(5-aziridinyl-3-hydroxymethyl-1methyl-2-[1H-indole-4,7-dione]prop-(3-en-a-ol),is however a much better substrate for NQO1 than MMC (Walton et al,1991) and a good correlation exists between NQO1 activity andchemosensitivity in vitro under aerobic conditions (Robertson et al,1994, Fitzsimmons et al, 1996, Smitkamp-Wilms et al, 1994). Underhypoxic conditions however, EO9's properties are markedly different withlittle or no potentiation of EO9 toxicity observed in NQO1 rich cells(Plumb and Workman, 1994). In NQO1 deficient cell lines however, largehypoxic cytotoxicity ratios have been reported (Workman, 1994).Therefore, EO9 has the potential to exploit the aerobic fraction of NQO1rich tumors or the hypoxic fraction of NQO1 deficient tumors (Workman,1994).

EO9 has been clinically evaluated but despite reports of three partialremissions in phase I clinical trials, no activity was seen againstNSCLC, gastric, breast, pancreatic and colon cancers in subsequent phaseII trials (Schellens et al, 1994, Dirix et al, 1996). These findings areparticularly disappointing in view of the preclinical studies (Hendrikset al, 1993) together with reports that several tumor types haveelevated NQ01 levels (Malkinson et al, 1992, Smitkamp-Wilms et al, 1995,Siegel et al, 1998). Several possible explanations have been proposed toexplain E09's lack of clinical efficacy (Connors, 1996, Phillips et al,1998). Recent studies have demonstrated that the failure of E09 in theclinic may not be due to poor pharmacodynamic interactions but may bethe result of poor drug delivery to tumors (Phillips et al, 1998). Therapid plasma elimination of E09 (tl/z=10 min in humans) in conjunctionwith poor penetration through multicell layers suggests that E09 willnot penetrate more than a few microns from a blood vessel within itspharmacokinetic lifespan (Schellens et al, 1994, Phillips et al, 1998).Intratumoural administration of E09 to NQ01 rich and deficient tumorsproduced significant growth delays (although a distinction betweendamage to the aerobic or hypoxic fraction was not determined) suggestingthat if E09 can be delivered to tumors, therapeutic effects may beachieved (Cummings et al, 1998b). While these undesirablecharacteristics are a serious setback for the treatment of systemicdisease, paradoxically they may be advantageous for treating cancerswhich arise in a third compartment such as superficial bladder cancer.In this scenario, drug delivery is not problematical via theintravesical route and the penetration of E09 into avascular tissue canbe increased by maintenance of therapeutically relevant drugconcentrations within the bladder (using a one hour instillation periodfor example). While this method of instilling EO9 within the bladder maybe useful, there still remains a need for drug delivery vehicles thatare capable of delivering an effective amount of EO9 within the bladder.

BRIEF SUMMARY OF THE INVENTION

In a broad aspect, the present invention is directed to compositions fortreating cancer. More specifically, the compositions of the presentinvention comprise pharmaceutical products formulated for intravesicalinstillation to treat bladder cancer. The pharmaceutical productscomprise bioredutive alkylating indoloquinone with anti-tumor effectssuch as, but not limited to,3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol(E09) and a formulation vehicle. The formulation vehicles of the presentinvention improves the physical characteristics of the solution such assolubility, lyophilization, and ease of reconstitution of thelyophilized solution.

According to one embodiment of the present invention, the composition ofthe present invention comprises3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol(E09) and a formulation vehicle. According to one embodiment, theformulation vehicle is a mixture of tert-butanol and water. In anotherembodiment, the formulation vehicle is a mixture of ethanol and water.In yet another embodiment, the formulation vehicle is2-hydroxypropyl-β-cyclodextrin. These composition embodiments of thepresent invention can be lyophilized by techniques known or developed inthe art. The lyophilized compositions of the present invention are

According to another embodiment of the present invention, thecomposition of the present invention comprises EO9 and a coating agent.The coating agent allows for better adhesion of the composition to thebladder wall. Consequently, the composition and, in particular, the EO9contacts and may be able to penetrate the avascular tissue thatcomprises for a time sufficient to treat the bladder cancer. In oneembodiment of the present invention, the coating agent is propyleneglycol. In other exemplary embodiments of the present invention, thecoating agent can be selected from the group consisting ofhydroxypropylcellulose, carboxymethylcellulose, chitosan hydrochloride,lectin, or polycarbophil. In yet another embodiment of the presentinvention, the compositions of the present invention can be delivered tothe bladder wall by a liposome. In another embodiment, the compositionsof the present invention can be delivered to the bladder wall by amicrosphere. In another embodiment, the compositions of the presentinvention can be delivered to a patient intravenously.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Validation of the polyclonal anti-rat NQO1 antibody for use inimmunohistochemical analysis of human NQO1. Panel A: Western blotanalysis of cell extracts (12.5 p,g protein loaded per lane) for NQO1.Lanes 1-5 represent extracts from DLD-1 (794±121 nmol/min/mg), HT-29(688±52 nmol/min/mg), H460 (1652±142 nmol/min/mg), MT1 (287±53nmol/min/mg), and RT112 (30±3 nmol/min/mg) respectively where the valuesin parenthesis represent NQ01 activity. Lane 6 represents molecularweight markers (ECL protein molecular weight markers, Amersham PharmaciaBiotech, UK). Panel B: Western blot analysis using purified humanrecombinant NQO1. Lanes 1-5 represent protein amounts of 0.25, 0.125,0.0625, 0.0312 and 0.0156 pmol respectively. Panel C: Western blotanalysis of cell extracts (25/,cg protein loaded per lane) derived fromH460 cells (lanes 1-2) and BE cells (lanes 3-4).

FIG. 2. Immunohistochemical localization of NQ01 in human bladdertumors, normal bladder, urethra and ureter. Tumors (panels A, B and C)were classified as G2 pTa (panel A, [×200]) and G3 pT2 (panels B [×100])and G3 pT4 (panel C [×200]) which had high to intermediate levels ofNQO1 activity as determined by biochemical methods. Panel D (×100)represents a histological section through a macroscopically normallooking section of bladder from a patient who underwent cystectomy for aG3a pT4 tumor; no tumor was identified in these sections but someinflammatory change was evident. Panels E and F (×200) represent urethraand ureter with no evidence of invasive or in situ carcinoma in thesesections. All sections have been stained with NQ01 antibody. Negativestaining (without primary antibody) were clear (data not shown).

FIG. 3. The relationship between NQO1 activity and the response of apanel of cell lines to E09 (panel A) or MMC (panel B) under normalphysiological pHe of 7.4 (o) or acidic pHe values of 6.0 ( ). Regressionanalysis data (as determined by Sigma Plot graphics) for E09 at pH 7.4were r=0.886, slope=−0.52 and at pH 6.0, regression analysis data forE09 was r=0.804 and slope=−0.51. For MMC, regression analysis at pH 7.4was r=0.849, slope=−0.19 and at pH 6.0, r=0.609, slope=−0.23.

FIG. 4. Response of HT-29 multicell spheroids following a one hourexposure to E09 under acidic (pHe=6.0, 0) and physiological (pHe=7.4, 0)extracellular pH conditions. Values presented are the means of 3independent experiments±standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the present invention are directed to compositionsfor treating bladder cancer via intravesical instillation. According toone embodiment, the composition of the present invention comprises3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol(EO9) and a formulation vehicle. The formulation vehicles of the presentinvention are solvents that improves the solubility and stability ofEO9. In a broad aspect of the present invention, the formulationvehicles of the present invention can be a mixture of an alcohol andwater. According to the various embodiments of the present invention,EO9 dissolves in the formulation vehicles without physical manipulationsuch as grinding. Because the compositions of the present invention arecapable of dissolving greater amounts of EO9, additional flexibilitywith respect to dosage units is achieved. According to one embodiment, acontent of 8.0 mg of EO9 per dosage unit is contemplated. In otherembodiments, instillation doses range from approximately 0.5 mg toapproximately 16 mg in a total volume of 40 mL.

In addition to improving the solubility of EO9, the formulation vehiclesof the present invention are good lyophilization vehicles. For example,the formulation vehicles of the present invention minimizes the time tolyophilize the compositions of the present invention. Accordingly, inone embodiment of the present invention, it is possible to lyophilizethe compositions of the present invention in less than approximately 4.5days. Furthermore, the compositions of the present invention are stableafter undergoing lyophilization (see table 4). It is believed that theformulation vehicles of the present invention minimize thecrystallization of EO9 during the lyophilization process. Consequently,by reducing the amount of crystallization of EO9, a smaller volume offluid is required to reconstitute the compositions of the presentinvention. As a result, a larger batch size can be achieved due to thereduced reconstitution volumes for the lyophilized composition.

According to one embodiment, the composition of the present inventioncomprises EO9 and a formulation vehicle comprising tert-butanol.According to another embodiment of the present invention, theformulation vehicle comprises mixture of ethanol and water. In yetanother embodiment, the formulation vehicle is2-hydroxypropyl-β-cyclodextrin. In one exemplary embodiment, theformulation vehicle comprises 40% tert-butanol in water. As thoseskilled in the art will appreciate, the amount of tert-butanol may bevaried. The tert-butanol solution better dissolves EO9 as compared towater. By utilizing a tert-butanol formulation vehicle, solubility ofEO9 is at least 9.5 mg/ml whereas the solubility of EO9 is approximately0.2 mg/ml in water. Consequently, a smaller volume of the tert-butanolis required to dissolve a given amount of EO9. Additionally, a greateramount of EO9 may be dissolved in a given solution. That is, thecompositions of the present invention will have a higher concentrationof EO9 as compared to a solution where EO9 is dissolved in water.

According to another embodiment of the present invention, thecomposition comprises, EO9, a formulation vehicle, and a bulking agent.In one exemplary embodiment, lactose can be utilized as the bulkingagent. As those skilled in the art will appreciate, it is contemplatedthat other bulking agents known or developed in the art may be utilized.According to another exemplary embodiment, the composition of thepresent invention can be buffered. In one embodiment, the composition isbuffered to a pH ranging from approximately 9 to approximately 9.5. Thecomposition can be buffered with any known or developed bufferingagents. The compositions of the present invention can either becompounded for intravesical delivery or lyophilized. As those skilled inthe art will appreciate, the compositions of the present invention canbe lyophilized by those methods known or developed in the art. Thelyophilized compositions can be reconstituted by a reconstitutionvehicle. According to one exemplary embodiment, the reconstitutionvehicle comprises 2% sodium bicarbonate, 0.02% disodium edetate andpropylene glycol: water (60:40 V/V). This reconstitution vehicledissolves the lyophilized composition of the present invention andproduces a stable solution for administration for up to 24 hours.Additionally, the reconstitution vehicle of the present inventionprovides an ampoule having an extractable volume of 5 mL ofreconstituted E)9 comprising propylene glycol/water/sodiumbicarbonate/sodium edetate 60/40/2/0.02% v/v/w/w.

In another aspect of the present invention, the compositions of thepresent invention also comprises coating agents. The coating agents ofthe present invention provide better adhesion of the composition to thebladder wall. Consequently, the composition and, in particular, the EO9contacts and may be able to penetrate the avascular tissue thatcomprises for a time sufficient to treat the bladder cancer. In oneembodiment of the present invention, the coating agent is propyleneglycol. In other exemplary embodiments of the present invention, thecoating agent can be selected from the group consisting ofhydroxypropylcellulose, carboxymethylcellulose, chitosan hydrochloride,lectin, or polycarbophil.

In yet another embodiment of the present invention, the compositions ofthe present invention can be delivered to the bladder wall by aliposome. According to one embodiment of the present invention, theliposomes used are unilamellar or multilamellar and contain at least onecationic phospholipid such as stearylamine,1,2-diacyl-3-trimethylammonium-propane (TAP) or1,2-triacyl-3-dimethylammonium-propane (DAP). In another embodiment ofthe present invention, the surface liposomes may be coated withpolyethylene glycol to prolong the circulating half-life of theliposomes. In yet another embodiment of the present invention, neutrallycharged liposomes such as, but not limited to, phosphatidylcholine andcholesterol can also be used for liposomal entrapment of thecompositions of the present invention. In another embodiment, thecompositions of the present invention can be delivered to the bladderwall by a microsphere such as those known or developed in the art.

In yet another embodiment, the compositions of the present invention canbe delivered to a patient intravenously. The lyophilized composition ofthe present invention can be reconstituted using the formulationvehicles of the present invention. The reconstituted composition canthen be diluted to a desired concentration and delivered to a patientintravenously.

The following experiments were conducted to determine the activity ofNQ01 in a series of human bladder tumors and normal bladder tissue byboth enzymatic and immunohistochemical techniques. Furthermore, thefollowing experiments evaluate strategies for reducing possible systemtoxicity arising from intravesical therapy based upon the fact that theaerobic activity of EO9 against cell lines is enhanced under mild acidicconditions (Phillips et al., 1992). Administration of EO9 in an acidicvehicle would result in greater activity within the bladder and any drugabsorbed into the blood stream would become relatively inactive due tothe rise in extracellular pH. The following experiments also determinethe role of NQ01 in the activation of EO9 under acidic conditions.

Collection of tumor and normal bladder specimens. Ethical approval fortissue collection was obtained from the Local Research Ethical Committee(Bradford NHS Trust) and samples taken from patients following informedconsent. A total of 17 paired cold pinch biopsies were taken frombladder tumors and macroscopically normal looking bladder mucosa atcystoscopy, immediately prior to formal transurethral resection of thetumor. Three specimens were taken from patients undergoing cystectomyand tumor and normal samples dissected by pathologists within one hourof surgical removal. Specimens were flash frozen in liquid nitrogen andtransported for NQOI enzyme analysis. Further biopsies were taken of thenormal bladder mucosa immediately adjacent to the previous biopsy siteand sent at the end of the procedure, along with the resected tumor, informalin for routine histological analysis. In this way bladder tumorand normal bladder urothelium enzymology could be directly correlatedwith the appropriate tissue histology in each patient.Immunohistochemistry was performed from the subsequently archived waxblocks prepared for histology.

Biochemical determination of NQOI activity. Cell cultures in exponentialgrowth were trypsinised, washed twice with Hanks balanced salt solution(HBSS) and sonicated on ice (3×30 sec bursts at 40% duty cycle andoutput setting 4 on a Semat 250 cell sonicator). NQO1 activity andprotein concentration was determined as described below. Tissues werehomogenised (10% w/v homogenate) in sucrose (0.25M) using a 1 ml tissuehomogeniser (Fisher Scientific). Cytosolic fractions were prepared bycentrifugation of the homogenate at 18,000 g for 4 min followed byfurther centrifugation of the supernatant at 110,000 g for 1 h at 4° C.in a Beckman Optima TL ultracentrifuge. Activity of NQO1 in thesupernatant was determined spectrophotometrically (Beckman DU650spectrophotometer) by measuring the dicumarol sensitive reduction ofdichlorophenolindophenol (DCPIP, Sigma Aldrich, UK) at 600 nm (Traver etal, 1992). This assay has been extensively validated for use inmeasuring NQO1 activity in both tissue and cell homogenates and has beenshown to be preferable to other assays for NQO1 activity (Hodnick andSartorelli, 1997). Each reaction contained NADH (200 IzM), DCPIP (40/iM,Sigma Aldrich, UK), Dicumarol (20 uM, when required, Sigma Aldrich, UK),cytosolic fraction of tissues (50 p,l per assay) in a final volume of 1ml Tris HCl buffer (50 mM, pH 7.4) containing bovine serum albumin (0.7mg ml⁻¹, Sigma Aldrich, UK). Rates of DCPIP reduction were calculatedfrom the initial linear part of the reaction curve (30s) and resultswere expressed in terms of nmol DCPIP reduced/min/mg protein using amolar extinction coefficient of 21 mNT′ cm⁻¹ for DCPIP. Proteinconcentration was determined using the Bradford assay (Bradford, 1976).

Immunohistochemistry. Polyclonal antibodies (raised in rabbits) topurified rat NQO1 were a gift from Professor Richard Knox (Enact PharmaPlc). Validation of the antibody for use in immunohistochemistry studieswas performed by Western blot analysis using both purified humanrecombinant NQO1 and cell extracts derived from a panel of cell lines ofhuman origin. These cell lines included H460 (human NSCLC), RT112 (humanbladder carcinoma), HT-29 (human colon carcinoma), BE (human coloncarcinoma), MT1 (human breast) and DLD-1 (human colon carcinoma). The BEcell line has been genotyped for the C609T polymorphic variant of NQOIand is a homozygous mutant (and therefore devoid of NQO1 enzymeactivity) with respect to this polymorphism (Traver et al, 1992). Cellswere washed in ice cold phosphate buffered saline and lysed bysonication (30 seconds on ice) in Tris HCl (50 mM, pH 7.5) containing 2mM EGTA, 2 mM PMSF and 25 Ftg ml⁻¹ leupeptin. Protein concentration wasestimated using the Bradford assay (Bradford, 1976) and a total of 12.5,ug of protein (in Lamelli sample loading buffer) applied to a 12%SDS-PAGE gel. Following electrophoretic transfer to nitrocellulosepaper, membranes were blocked in TBS/Tween 20 (0.1%) containing 5%non-fat dry milk for 1 h at room temperature. Membranes were washed inTBS/Tween 20 (0.1%) prior to the addition of rabbit anti-rat NQO1antibody (1:100 dilution) and incubated at room temperature for 1 h.Membranes were extensively washed in TBS/Tween 20 (0.1%) followed by theaddition of anti-rabbit IgG horseraddish peroxidase conjugated secondaryantibody (1:5000 dilution in TBS/Tween 20). Proteins were visualised byECL based chemiluminescence as described by the manufacturer (AmershamPharmacia Biotech, Bucks, UK).

For immunohistochemical studies, all tissues (both tumor and normalbladder mucosa) were fixed in 10% formalin, processed routinely andembedded in paraffin wax. Two sections of each tissue block were placedon one slide, one section served as the test and the other as a negativecontrol (no primary antibody). A total of 5 sections from each samplewere stained for NQ01 (plus negative controls) and tumor and normalsamples from a total of 17 patients were analysed. Sections (5, um) weredewaxed, rehydrated and incubated with primary antibody (1:400 dilution)for 4 hours. Sections were then washed and incubated with biotinylatedmouse anti rabbit IgG for 30 min prior to immunoperoxidase stainingusing VECTASTAIN ABC reagents and DAB (Vector Laboratories Ltd,Peterborough, UK). Sections were counterstained with haematoxylinaccording to standard procedures.

Cell culture and chemosensitivity studies. E09 was a gift from NDDOOncology, Amsterdam and MMC was obtained from the Department ofPharmacy, St Lukes Hospital, Bradford. H460 (human NSCLC) cell line wasobtained from the American Type Culture Collection (ATCC). HT-29 (humancolon carcinoma), RT112/83 (human bladder carcinoma epithelial), EJ138(human bladder carcinoma) and T24/83 (human bladder transitional cellcarcinoma) cell lines were obtained from the European Collection ofAnimal Cell Cultures (ECACC). A2780 (human ovarian carcinoma) and BE(human colon carcinoma) cells were gifts from Dr T Ward (PatersonInstitute, Manchester, UK). All cell lines were maintained as monolayercultures in RPNII 1640 culture medium supplemented with fetal calf serum(10%), sodium pyruvate (2 mM), L-glutamine (2 mM),penicillin/streptomycin (50 IU/ml/50 jug/ml) and buffered with HEPES (25mK. All cell culture materials were purchased from Gibco BRL (Paisley,UK). Cells were exposed to MMC or E09 at a range of doses for one hourand chemosensitivity was assessed following a five day recovery periodusing the MTT assay, details of which have been described elsewhere(Phillips et al, 1992). The pH of the medium used during drug exposurewas adjusted using small aliquots of concentrated HCl (40, A conc HCl[10.5M] to 20 ml medium gives a pH of 6.0). Calibration curves wereconducted over a broad range of pH values in culture medium (pH 3.5 to11) and the stability of the pH conditions monitored over a one hourincubation period at 37° C. At all pH values, no significant changes inthe pH of the medium was observed over the one hour drug exposure period(data not presented).

HT-29 multicell spheroids were prepared by seeding 5×10⁵ cells into T25flasks which had been based coated with agar (1% w/v) and incubated for24 h at 37° C. Immature spheroids were then transferred to a spinnerflask (Techne) containing 250 ml of RPMI 1640 growth medium andspheroids were kept in suspension by stirring at 50 rpm. When spheroidsreached a diameter of approximately 500 Am, they were harvested forchemosensitivity studies. Multicell spheroids were exposed to a range ofE09 concentrations at pHe 6.0 and 7.4 for one hour at 37° C. Followingdrug incubation, spheroids were washed twice in HBSS prior todissagregation into single cells using trypsin EDTA. Disaggregatedspheroids were then washed in HBSS and then plated into 96 well plates(1×10³ cells per well). and incubated at 37° C. for four days.Chemosensitivity was assessed using the NM assay as described elsewhere(Phillips et al, 1992).

The role of NQO1 in the activation of E09 at pHe values of 7.4 and 6.0was evaluated using the NQO1 inhibitor Flavone Acetic Acid (FAA),details of which are described elsewhere (Phillips, 1999). FAA is acompetetive inhibitor of NQ01 with respect to NADH and at a finalconcentration of 2 mM, inhibition of NQO1 is >95% whereas the activityof cytochrome P450 reductase and cytochrome b5 reductase is notsubstantially altered (<5% inhibition). Briefly, H460 cells (NQO1 rich)were plated into 96 well plates at a density of 2×10³ cells per well.Following an overnight incubation at 37° C., medium was replaced withfresh medium (pH 7.4) containing a non-toxic concentration of FAA (2 mM)and incubated for one hour at 37° C. Medium was then replaced with freshmedium containing E09 (range of drug concentrations) and FAA (2 mM) ateither pHe 7.4 or 6.0. Following a further one hour incubation at 37°C., cells were washed twice with HBSS and incubated at 37° C. in growthmedium for five days. Chemosensitivity was determined by the NM assay asdescribed above and results were expressed in terms of IC5₀ values,selectivity ratios (IC_(So) at pHe 7.4/IC50 at pHe 6.0) and protectionratios (ICSO FAA/E09 combinations/IC50 for E09 alone).

Substrate specificity. The influence of acidic pHe on substratespecificity for purified human NQ01 was determined as describedpreviously (Phillips 1996, Walton et al, 1991). NQ01 mediated reductionof the quinone to the hydroquinone species is difficult to detect byconventional assays thereby necessitating the use of a reporter signalgenerating step. In this assay, the hydroquinone acts as an intermediateelectron acceptor which subsequently reduces cytochrome c which canreadily be detected spectrophotometrically. Recombinant human NQ01 wasderived from E. coli transformed with the pKK233-2 expression plasimdcontaining the full length cDNA sequence for human NQ01 isolated fromthe (Beall et al, 1994). Following IPTG induction, NQO1 was purified bycybacron blue affinity chromatography, details of which are describedelsewhere (Phillips, 1996). The purified protein had a molecular weightof approximately 31 kDa and a specific activity of 139/Amol DCPIPreduced/min/mg protein (Phillips, 1996). Reduction of E09 by recombinanthuman NQO1 was determined at pH 6.0 and 7.4 by measuring the rate ofreduction of cytochrome c was measured at 550 nm on a Beckman DU 650spectrophotometer according to previously published methods (Phillips,1996). Results were expressed in terms of, umol cytochrome creduced/min/mg protein using a molar extinction coefficient of 21.1mM⁻¹cm⁻¹ for cytochrome c.

Measurement of intracellular pH. Intracellular pH was determined usingthe fluorescent pH indicator BCECF (2,7-bis-(2-carboxy-ethyl)-5-(and-6)carboxyfluorescein (Molecular Probes, Eugene, USA) according tomanufacturers instructions. Confluent flasks of cells were washed withHBSS to remove any traces of serum containing RPMI medium and thenincubated with the esterified form of BCECF (BCECF-AM) at aconcentration of 2 [tM in HBSS for one hour at 37° C. The non-denaturingdetergent Pluronic was added to the probe to aid dispersion. Cells werethen washed to remove all traces of BCECF-AM and then trypsinized beforebeing suspended in serum-free/phenol red-free RPM1 medium (Gibco BRL,Paisley, UK) at a concentration of 10⁶ cells per ml at pH 6 for onehour. Flourescence measurement was determined in a Perkin-Elmerfluorescence spectrophotometer in UV grade disposable 4 ml cuvettes(Fischer Scientific) with excitation wavelengths 500 nm and 450 nm(excitation bandpass slit of 10 nm) and emission wavelength fixed at 530nm (emission bandpass slit of 2.5 nm). These were determined to beoptimal settings for the machine and system under study. An in-situcalibration was performed for every pHi determination with a range ofsix pH's from 4 to 9 using the ionophore nigericin at a concentration of22.8 p,M to equilibrate pHe with pHi. Calculation of the ratio offluorescence at 500 nm/450 nm was calculated after subtraction ofbackground fluorescence from blanks at each pH (serum free, phenol redfree RPMI without cells).

Activity of NQO1 in tumor and normal bladder specimens. The biochemicalactivity of NQO1 in paired samples of tumor (grade/stage ranging from G2pTa to G2/G3 T4) and normal bladder mucosa (with three cystectomyspecimens) taken from a series of 20 patients is presented in table 1.Within the tumor specimens, a broad range of NQO1 activity existedranging from 571.4 nmoUmin/mg to undetectable (<0.1 nmol/min/mg). Inhistologically normal bladder mucosa specimens, NQO1 activity rangedfrom 190.9 to <0.1 nmoUmin/mg. In the majority of patients NQO1 activityin the tumor was greater than in the normal bladder mucosa. Tumor gradeand stage did not correlate with NQO1 activity (table 1).

Validation of NQO1 antibody and immunohistochemical localization ofNQ01. Western blot analysis demonstrates that polyclonal anti rat NQO1antibody cross reacts with human NQO1 (FIG. 1) with a single band atapproximately 31 kDa observed for both cell extracts and purified humanNQO1. Titration of purified NQO 1 results in a decrease in bandintensity (FIG. 1B) and in cell extracts, band intensity wasqualitatively consistent with NQO1 enzyme activity (FIG. 1A). Inaddition, the antibody does not detect NQO1 in the BE cell line which isdevoid of NQ01 activity as a result of the C609T polymorphism (FIG. 1C).No non-specific bands were observed on Western blots. Immunoperoxidasestaining of NQO1 protein in tumor tissue, bladder wall, ureter andurethra are presented in FIG. 2. Superficial and invasive tumors(pTa—panel A, G3 pT2—panel B and G3pT4—panel C) with high tointermediate levels of NQO1 as determined by biochemical assays (patientnumbers 1, 4 and 5 in table 1) clearly stained positive for NQO1.Staining was confined to the cytoplasm of tumor cells with little or nostaining of stromal cells (panels B and C).

In other tumors with intermediate or low levels of NQOI activity,staining was heterogeneous with pockets of cells containing high levelsof NQ01 protein (data not shown). Normal bladder wall sections wereobtained from a patient who underwent cystectomy (G3pT4 bladder tumor),ureter and urethra were obtained from another patient who underwentcystectomy (G3 pT3a bladder tumor). In the bladder wall, no NQO1staining was observed in the urothelium (panel D) although slightstaining was present in smooth muscle layers. The urethra (panel E) wasnegative although cells on the luminal surface of the ureter werepositively stained (panel F). The basal layers of the ureter lining werehowever negatively stained (panel F). No evidence of invasive malignancyor in situ carcinoma were observed in the ureter and urethra or in thesection of bladder wall presented (panel D). In 16 other normal bladderbiopsy and cystectomy specimens, no positive staining of the urotheliumwas observed (data not shown).

Influence of pH on substrate specificity and chemosensitivity. Theability of E09 to serve as a substrate for NQO1 was not influenced by pHwith specific activities of 21.10±2.3 and 21.30±1.5 pmol cytochrome creduced/min/mg protein at pH 7.4 and 6.0 respectively. The response of apanel of cell lines with a range of NQO1 activity (<1.0 to 1,898±276nmol/min/mg) to E09 and MMC at pHe values of 7.4 and 6.0 is presented intable 2 and FIG. 2. At pHe=7.4, a good correlation existed between NQ01activity and chemosensitivity to E09 (FIG. 3). In the case of MMC (table2, FIG. 3), a relationship between NQ01 and chemosensitivity wasapparent (at pHe 7.4) although this relationship was not as prominent asshown by E09 with a narrow range of IC50 values (range 0.9 to 7.0 ttM)observed in cell lines which cover a broad range of NQ01 activity(ranging from <1.0 to 1,898 nmol/min/mg). Both MMC and E09 arepreferentially more toxic to cells at pHe values of 6.0 although muchgreater potentiation of E09 activity is seen with SR values(SR=selectivity ratio defined as IC₅₀ pHe 7.4/IC50 pHe 6.0) ranging from3.92 to 17.21 for E09 compared with 1.02 to 4.50 for MMC (table 2). Theactivity of E09 was enhanced in both NQO1 rich and deficient cell lineswhen pHe was reduced to 6.0 and the relationship between NQO1 andchemosensitivity remained good when cells were exposed to E09 underacidic conditions (FIG. 3). No cell kill was observed in controlcultures when the pHe was decreased to 6.0 (in the absence of drug) asdetermined by the MTT assay. The response of H460 cells to E09 at pHevalues of 7.4 and 6.0 in the presence and absence of FAA (2 mM) ispresented in table 3. At both pHe values, the response of H460 cells toE09 was reduced in the presence of FAA. Protection ratios defined as theIC50 for E09 plus FAA divided by the IC50 value for E09 alone weresimilar for cells under acidic and physiological pHe values (14.63 and13.95 respectively, table 3). Selectivity ratios defined as the IC50 atpHe 7.4 divided by the IC50 at pHe 6.0 in the presence and absence ofFAA were also similar with SR values of 6.31 and 6.02 for E09 alone andE09 plus FAA respectively (table 3). The response of HT-29 multicellspheroids to E09 is presented in FIG. 4. Spheroids exposed to E09 at pHe6.0 were significantly more responsive than at pHe 7.4 with IC5o valuesof 9.89±0.89 and 24.24±3.29 AM respectively. Spheroids weresignificantly less responsive to E09 than the same cells exposed to E09as monolayers at both pHe values with ratios of IC50 values forspheroids to monolayers of 202 and 341 at pHe values of 7.4 and 6.0respectively.

Influence of acidic pHe conditions on pHi. PM values following a onehour incubation at pHe 6.0 were 6.44±0.04, 6.51±0.02 and 6.42±0.05 inA549, RT112/83 and A2780 cells respectively. Addition of the ionophorenigericin (after a one hour incubation at pHe 6.0) resulted in theequilibration of pHe and p11i.

In terms of bioreductive drug development, two of the critical factorswhich will ultimately determine selectivity are the enzymology of tumorsand the presence of hypoxia (Workman, 1994). As outlined in theintroduction, the presence or absence of NQ01 is central to the designof appropriate E09 based therapeutic strategies aimed at targetingeither the aerobic (NQO1 rich cells) or hypoxic fraction (NQO1 deficienttumors) of tumors. Workman (1994) has outlined a proposed mechanism forthe different properties of E09 under aerobic and hypoxic conditionsbased on the hypothesis that it is the semiquinone (product of oneelectron reduction) rather than the hydroquinone which is responsiblefor toxicity. In NQO1 deficient cells, the semiquinone produced as aresult of one electron reductases would be relatively non toxic as itwould rapidly redox cycle back to the parent compound. Free radicalspecies generated as a result of redox cycling would be detoxified bysuperoxide dismutase or catalase but under hypoxic conditions, thesemiquinone would be relatively stable. If this were the major toxicspecies, then the activity of E09 against cells with low NQO1 would bepotentiated. In NQO1 rich cells however, the major product formed wouldbe the hydroquinone. Aerobic toxicity could be generated as a result ofthe back oxidation of the hydroquinone to the semiquinone species or theparent quinone (Butler et al, 1996) resulting is free radicalgeneration. Under hypoxic conditions however the hydroquinone will bemore stable and if this is relatively nontoxic, then the activity of E09against NQO1 cells under hypoxia would not be potentiated. Whilst themechanism of action of E09 under aerobic and hypoxic conditions iscomplex, the biological data suggest that E09 should target the aerobicfraction of NQO1 rich tumors or the hypoxic fraction of NQO1 deficienttumors (Workman, 1994).

Analysis of NQ01 activity in tumor and normal bladder tissues hasclearly identified patients whose tumors are either NQO1 rich or NQO1deficient (table 1). Within the subset of NQ01 rich tumors, enzymeactivity is elevated relative to the normal bladder urothelium.Immunohistochemical studies confirm these biochemical measurements withstaining confined to tumor cells as opposed to normal stromal cells(FIG. 2, panels A, B and C). Within normal bladder tissues, NQ01staining was absent from the urothelial lining of the bladder (FIG. 2,panel D) and the urethra (FIG. 2, panel E). Faint staining of thesuperficial layers of the ureter (FIG. 2, panel F) was observed althoughthe underlying basal layers of the ureter were negatively stained.Similarly, faint staining of the smooth muscle layers of the bladder,ureter and urethra were also observed (data not shown). These studiessuggest that a proportion of patients with bladder tumors (at variousgrades and stages of the disease) exhibit a significant differential interms of NQO1 activity which could potentially be exploited by E09 basedtherapies directed against the aerobic fraction of tumor cells. Withregards to the ability of E09 to selectively kill hypoxic NQ01 deficientcells, a subset of patients also exist whose tumors are devoid of NQ01activity (table 1). It is not known whether or not bladder tumorscontain regions of low oxygen tension and further studies are requiredusing hypoxia markers such as pimonidazole (Kennedy et al, 1997) toaddress this issue and to establish the relationship between NQ01activity and hypoxia in tumors.

Whilst biochemical and immunohistochemical studies demonstrate that asubset of patients exist which have the appropriate tumor enzymology toactivate E09 (under aerobic conditions), intravesical chemotherapy canresult in systemic toxicity due to the drug entering the blood supply.This study has also evaluated a potential strategy for minimizing anyrisk of systemic toxicity based upon the hypothesis that administrationof E09 in an acidic vehicle would enhance the potency of E09 (Phillipset al, 1992) within the bladder and that any drug reaching the bloodstream would become relatively inactive due to a rise in pHe.Selectivity for aerobic cells would still be determined by NQO1 activityand therefore it is essential to determine the role that NQO1 plays inthe activation of E09 under acidic pHe conditions. In a panel of celllines with a broad spectrum of NQO1 activity, reducing the pHe to 6.0enhances the potency of E09 under aerobic conditions in all cases (withSR values ranging from 3.92 to 17.21, table 2). In the case of MMC,potency is also enhanced at low pHe values although the magnitude of thepH dependent increase in toxicity is reduced (SR values ranging from1.02 to 4.50, table 2) compared with E09. With respect to MMC, oneexplanation for increased activity under acidic conditions has beenattributed to the fact that MMC becomes a substrate for NQO1 underacidic conditions (Pan et al, 1993, Siegel et al, 1993). This is not thecase with E09 as rates of reduction of E09 by purified human NQO1 arenot influenced by pH (21.10±2.30 and 21.30±1.50 limol cytochrome creduced/min/mg protein at pH 7.4 and 6.0 respectively). Recent studieshave demonstrated that the activity of E09 is enhanced under acidicconditions (pHe=6.5) but only when the intracellular pH is reduced(plli=6.5) by co-incubation with nigericin (Kuin et al, 1999). Theresults of this study are in agreement with this finding as pHi becomesacidic (pHi values range from 6.42±0.05 to 6.51±0.02 depending on thecell line) when cells are cultured under pHe 6.0 conditions.

In the panel of cell lines used in this study, a good correlation existsbetween NQO1 activity and chemosensitivity at both pHe values of 7.4 and6.0 (FIG. 3). A strong relationship between NQO1 activity and responseunder aerobic conditions (at pHe 7.4) has been established previously byseveral groups (Robertson et al, 1994, Fitzsimmons et al, 1996,Smitkamp-Wilms et al, 1994) and there is clear evidence that NQ01 playsa central role in the mechanism of action of E09 under aerobicconditions (Workman, 1994). The good correlation between NQ01 activityand response at pHe 6.0, in conjunction with the fact that E09 is stilla good substrate for NQ01 at pH 6.0, suggests that NQO1 plays asignificant role in E09's mechanism of action at acidic pHe values underaerobic conditions. It is of interest to note however that the activityof E09 against BE cells (which are devoid of NQ01 activity as a resultof the C609T polymorphism, Traver et al, 1992) is also enhanced underacidic pHe conditions (table 2). This suggests that there is a NQO1independent mechanism for the increased activity of E09 under acidicconditions. This is confirmed by the use of the NQ01 inhibitor FAA wherethe ‘protection ratios’ (defined as the ratio of IC₅₀ values for E09plus FAA divided by the ICSo values for E09) are similar at both pHe 7.4and 6.0 (13.95 and 14.63 respectively, table 3). If NQO1 played acentral role in the activation of E09 at pHe 6.0, then the protectionratio at pHe 6.0 would be significantly greater than the protectionratio at pHe 7.4. The mechanism behind the NQ01 independent activationof E09 is unclear although it is a well known fact that the reactivityof aziridine ring structures is enhanced by protonation resulting inring opening to the aziridinium ion which is a potent alkylating species(Mossoba et al, 1985, Gutierrez, 1989). Alternatively, E09 is asubstrate for other one electron reductases (Maliepaard et al, 1995,Saunders et al, 2000) and further studies designed to evaluate whetherE09's metabolism by these enzymes is pH dependent needs to bedetermined. The potency of E09 can be enhanced further by reducing pHebelow 6.0 (Phillips et al, 1992) but these conditions are unlikely toprovide significant clinical benefits as E09 becomes progressively moreunstable when pH is reduced to 5.5 (t'/s=37 min). From a pharmacologicalstandpoint, administration of E09 in a vehicle at pH 6.0 would appeardesirable. Not only would this result in significant enhancement of E09activity but also the stability of E09 would be sufficient (tIh=2.5 h)to maintain drug exposure parameters at a therapeutic level.

With regards to the activity of E09 against three dimensional culturemodels in vitro, this study has demonstrated that reducing the pHe to6.0 enhances the potency of E09 against multicell spheroids although themagnitude of this effect is reduced compared with monolayer cultures(FIG. 4). It is not known whether or not reduction in pHe results ingreater cell kill throughout the spheroid or if it is confined to thesurface of the spheroid exposed to medium. In comparison with MMC,previous studies using histocultures exposed to MMC demonstrated that nodifference in toxicity exists between physiological and acidic pHeconditions (Yen et al, 1996). The pH dependent increase in E09 toxicityagainst spheroids suggests that manipulation of pHe may not only be ofuse in treating a multilayered solid bladder tumor but may offer anadvantage over MMC. It should however be stated that multicell spheroidsare significantly less responsive to E09 than monolayers, presumablybecause of the poor penetration properties of E09 through avasculartissue (Phillips et al, 1998). E09 can nevertheless kill >90% of cellsin spheroids (FIG. 4) suggesting that a higher doses at least, thepenetration of E09 is sufficient to eradicate cells which reside somedistance away from the surface of the spheroid.

In conclusion, the results of this study have demonstrated that within apopulation of patients with bladder tumors at various stages and gradesof the disease, there exists a great heterogeneity regarding theexpression of NQO1. The majority of patients have tumors possessingelevated levels of NQO 1 while a small subset of patients appear to bedevoid of NQO1 activity. The heterogeneous nature of NQO1 activitydescribed here is consistent with several other studies in various tumortypes (Malkinson et al, 1992, Smitkamp-Wilms et al, 1995, Siegel et al,1998). These findings reinforce the view that ‘enzyme profiling’ ofindividual patients could be valuable prior to therapeutic interventionwith bioreductive drugs (Workman, 1994). This is to our knowledge thefirst study to characterize NQO1 activity and cellular localization inbladder tumors and provide strong evidence to support the evaluation ofE09 against superficial and locally invasive bladder tumors. This studyhas clearly demonstrated that under aerobic conditions, E09 is much morepotent under acid conditions (pH6.0) than at physiological pH (pH7.4).The mechanism for this increased E09 potency appears to be NQ01independent and whilst this will not improve (or reduce) selectivity, itmay prove beneficial in terms of reducing the therapeutically effectivedose of E09. Dose reduction in conjunction with the fact that areduction in the potency of E09 due to the increased pHe in the bloodstream suggests that systemic toxicity arising from the intravesicaladministration of E09 would be low. In addition, this study shows thatunder physiological conditions the activity of E09 is much lower intissues with “normal” expression of NQO1 compared to “high” NQO1expressing tissues (i.e. the tumors). The results of this study providestrong evidence in support of the proposal that intravesicaladministration of E09 may have activity against bladder tumors.

References

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TABLE 1 Tumor histology reports and NQO1 activity in paired samples ofbladder tumor and normal bladder mucosa. NQO1 Activity Ratio of NQO1Activity Normal NQO1 levels Patient Tumor Tumor (nmol/ in tumor to No.histology (nmol/min/mg) min/mg) normal tissue.  1 ^(f,s,i,p) G2 pTa571.4 <0.1 571.  2 ^(m,s,r) G3 pT2 273.3 <0.1 273.  3 ^(f,s,i) G1pTa107.80 <0.1 107.  4 ^(m,e,i) G3 pT2/3 73.36 <0.1 73.3  5 ^(m,s,i) G3pT4(0′ 81.30 4.10 19.8  6 ^(h) G2PT1 309.50 25.20 12.1  7 ^(m,n,r,o) G3 pT210.00 <0.1 10.0  8 ^(fn,i) G3pT2 9.80 <0.1 9.80  9 m,n,i G2 pT2 4.40<0.1 4.40 10 m,s,c G3 pT2 34.01 8.50 4.00 11 ^(m,s) G 1 pTa 69.76 22.203.14 12 ,,n G1pTa 42.16 15.30 2.73 13 m,n,i G3 pT2 179.6 72.12 2.49 14m,e,i G2/G3 T4 (C) 89.70 63.30 1.41 15 m,n,r G3 pT2 0.40 <0.1 0.40 16m,e,c,o G3 PT3 (C) 21.60 61.70 0.35 17 f n,i G2 PTI 58.40 190.90 0.30 18m,e,o G2 PTI <0.1 <0.1 0 19 f n,i G2 PTI. <0.1 <0.1 0 20 m,e,c,r G2 pT0<0.1 <0.1 0 ^(m) Male, ^(f) Female,^(s) Smoker, ′Non-smoker, ^(e)Ex-smoker, ^(,,) Intravesical chemotherapy prior to specimen collection,^(r) Radiotherapy prior to specimen collection,^(i) First presentation,P Previous malignancy other than bladder, ^(h) No medical historyavailable, ^(o) Possible occupational # carcinogen exposure (i.e., dyeindustry worker). (C) denotes cystectomy specimens. In all cases,protein levels following preparation of the cytosolic fraction weregreater than 0.1 mg/ml.

TABLE 2 The relationship between NQ01 activity and chemosensitivity toE09 and MMC under physiological and acidic pHe conditions. IC50 pHe IC50pHe NQ01 7.5 6.0 Cell line Drug (nmol/min/mg) (nM) (nM) SR* H460 E091652 ± 142  60 ± 10 9.5 ± 2   6.31 HT-29 E09 − 688 ± 52 120 ± 53 29 ± 104.13 T24/83 E09 285 ± 28 290 ± 65 60 ± 18 4.83 A2780 E09 159 ± 33 200 ±50 51 ± 14 3.92 EJ138 E09  83 ± 14 310 ± 95 39 ± 7  7.94 RT112 E09 30 ±3 1050 ± 75  61 ± 13 17.21 BE E09 <0.1 5300 ± 169 1300 ± 75−    4.07H460 MMC 1652 ± 142  900 ± 200 220 ± 130 4.50 HT-29 MMC 688 ± 52 1050 ±210 500 ± 240 2.10 T24/83 MMC 285 ± 28 2150 ± 93  2100 ± 800  1.02 A2780MMC 159 ± 33 2400 ± 340 1400 ± 130  1.71 EJ138 MMC  83 ± 14 1600 ± 2001400 ± 250  1.14 RT112 MMC 30 ± 3 3350 ± 250 2000 ± 500  1.67 BE MMC<0.1 7000 ± 192 4400 ± 215  1.59 All results presented are the mean of 3independent experiments (SD values omitted in the interests ofpresentation). *SR (selectivity ratio) = IC5o at pH 7.4/IC5o at pH 6.0

TABLE 3 Response of H460 cells to E09 in the presence or absence of FAA(2 mm) at pHe values of 7.4 and 6.0. ICso Drug pHe (nM) SR* PR** E09 7.560.0 ± 8.1 ′ — E09 6.0  9.5 ± 2.6 6.31 — E09/FAA 7.4 837 ± 45 — 13.95E09/FAA 6.0− 139 ± 27 6.02 14.63 *SR = Selectivity Ratio defined as theratio of ICSo values at pHe = 7.4 divided by the IC₅₀ at pHe = 6.0.**Plf = Protection ratio defined as the ratio of IC5_(o) values for E09plus FAA divided by the IC5o values for E09 alone. All values representthe mean ± standard deviation for three independent experiments.

TABLE 4 Neoquin 8 mg/vial lyophilised product time (months) Storage testitem 0 1 2 3 6  5° C. content* 102.7 ± 1.2  na na 103.8 ± 0.8  100.6 ±0.6  purity**  99.9 ± 0.008 na na 99.5 ± 0.03 99.6 ± 0.03 residual 6.0%na na 7.0% 6.3% moisture*** pH after 9.5    na na na 9.4   reconstitution**** 25° C./60% content 102.7 ± 1.2  103.4 ± 0.7  102.1 ±0.2  102.6 ± 1.3  97.4 ± 1.0 RH purity  99.9 ± 0.008 99.9 ± 0.05 99.9 ±0.01 99.2 ± 0.07 98.7 ± 0.2 residual moisture 6.0% na na 5.9% 5.9% pHafter 9.5    na na na 9.4    reconstitution**** 40° C./75% content 102.7± 1.2  102.3 ± 1.1  100.4 ± 1.3  101.3 ± 0.2  86.4 ± 2.0  RH purity 99.9 ± 0.008 99.8 ± 0.01 99.7 ± 0.04 98.4 ± 0.07 97.5 ± 0.2  residualmoisture 6.0% na na 6.2% 6.3% pH after 9.5    na na na 9.5   reconstitution**** *content as % of labelled content n = 3 **purity aschromatographic purity n = 3

1. A stabilized anti-cancer formulation comprising:3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]propenoldissolved in tert-butanol/water having a pH ranging from approximately 9to approximately 9.5, and a bulking agent, wherein said stabilizedanti-cancer formulation is lyophilized.
 2. The stabilized anti-cancerformulation of claim 1, wherein the formulation is for treating bladdercancer and said bulking agent is lactose.
 3. The stabilized anti-cancerformulation of claim 1, further comprising a reconstitution vehicle,wherein the formulation is for treating bladder cancer.
 4. Thestabilized anti-cancer formulation for treating bladder cancer of claim3 wherein said reconstitution vehicle is a solution comprising 2% sodiumbicarbonate, 0.02% disodium edentate and propylene glycol:water (60:40v/v).
 5. A stabilized anti-cancer formulation produced by: admixing3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]propenoldissolved in a formulation vehicle having a pH ranging fromapproximately 9 to approximately 9.5, and a bulking agent, wherein theformulation vehicle is tert-butanol/water or a solution of2-hydroxypropyl-β-cyclodextrin; and lyophilizing.
 6. The stabilizedanti-cancer formulation produced by the process of claim 5, wherein saidformulation vehicle is tert-butanol/water.
 7. The stabilized anti-cancerformulation produced by the process of claim 5, wherein said bulkingagent is lactose.
 8. A stabilized anti-cancer formulation consistingessentially of:3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]propenoldissolved in tert-butanol/water having a pH ranging from approximately 9to approximately 9.5, and a bulking agent, wherein said stabilizedanti-cancer formulation is lyophilized; and a reconstitution vehicle. 9.The stabilized anti-cancer formulation of claim 8 wherein saidreconstitution vehicle is a solution consisting essentially of 2% sodiumbicarbonate, 0.02% disodium edentate and propylene glycol:water (60:40v/v).
 10. An anti-cancer composition comprising: a lyophilized productof a formulation consisting of3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]propenoldissolved in tert-butanol/water having a pH ranging from approximately 9to approximately 9.5, and lactose, reconstituted in a reconstitutionvehicle consisting of 2% sodium bicarbonate, 0.02% disodium edentate andpropylene glycol:water (60:40 v/v).